The present invention relates to processes for the preparation of a solution comprising a substantially pure isoform of AT-III, said process comprising separating the isoform AT-IIIxcex1 from AT-IIIxcex2 on a calcium hydroxyphosphate-based adsorbent.
Antithrombin III (AT-III) is a plasma glycoprotein that inhibits serine proteases in the coagulation cascade and thus plays a major role in the regulation of blood clotting. Antithrombin III is an inhibitor of Factors IXa, Xa, XI, XIIa, and thrombin. Thus, AT-III regulates clot formation in different stages of the coagulation cascade. A small decrease of the AT-III content in the blood is associated with increased risk of thromboembolism. AT-III concentrates are used in the prophylaxis and treatment of thromboembolic disorders in patients with acquired or hereditary antithrombin deficiency. In addition, it has been reported that AT-III is involved in many other biological responses, for example angiogenesis and inflammatory responses. The function of AT-III in these mechanisms is not yet fully understood.
Purification of AT-III with affinity chromatography, using heparin as the solid phase bound ligand, is known in the art. Miller-Andersson et al. (Thrombosis Research 5, 439-452, 1974) discloses the use of heparin-Sepharose to purify human AT-III. The entire procedure, which included ion exchange and gel filtration chromatography, provided a 34% yield.
In human plasma, antithrombin III exists as at least two molecular entities, which are homologous according to amino acid composition, but differ in carbohydrate content and in their heparin-binding behavior. An antithrombin variant, designated as AT-IIIxcex2, was isolated from human plasma independently from the predominant antithrombin species (designated as AT-IIIxcex1), by virtue of its tight binding to a heparin-Sepharose matrix at high ionic strengths (Peterson, C. B. and Blackburn, M. N. (1985) J. Biol. Chem. 260, 610-615).
The determined molecular weights were 59,800 and 56,900 for human AT-IIIxcex1 and AT-IIIxcex2, respectively. The difference in molecular weights of the two antithrombins was attributed to a reduction of approximately 25-30% in the sialic acid, neutral sugar, and amino sugar content of AT-IIIxcex2 when compared to the carbohydrate content of the AT-IIIxcex1 subspecies (Peterson and Blackburn, supra). It has been shown that AT-IIIxcex2 lacks one of the four oligosaccharide side-chains, namely the side-chain at asparagine 135 (Brennan, S. O. et al. (1987) FEBS Letters 219, 431-436). The AT-IIIxcex1 form is more negatively charged than AT-IIIxcex2; it has been demonstrated that AT-IIIxcex1 and AT-IIIxcex2 have pI:s of 4.9 and 5.1, respectively (Frebelius, S. et al. (1996) Arteriosclerosis, Thrombosis, and Vascular Biology 16:1292-1297).
It is desirable to obtain pure AT-IIIxcex2, as this form has specific effects on the coagulation in the vessel wall. It has been shown that AT-IIIxcex2 can prevent restenosis of the rabbit aorta after balloon injury (Swedenborg (1998) Blood Coagulation and Fibrinolysis 9 (suppl. 3):S7-S10). AT-IIIxcex2 may therefore be considered as a potential drug for humans in prophylaxis of restenosis when performing balloon dilatation of the aorta.
Histidine-rich glycoprotein (HRGP) is a single-chained plasma protein originally isolated in 1972. The exact physiological function of HRGP is still unknown. Due to interaction with heparin, fibrinogen and fibrin, plasminogen and activated platelets, HRGP is considered to be a modulator of coagulation and fibrinolysis (Koide, T. In: Fibrinolysis: Current Prospects. Gaffney, PJ (Ed.), John Libbey and Co., London 1988, p.55-63). The polypeptide chain consists of 507 amino acid residues and contains regions that share homology with other plasma proteins, e.g. antithrombin-III (Koide, T. et al. (1986) Biochemistry 25, 2220-2225).
As indicated above, the complete involvement of the two AT-III isoforms and HRGP in the body is not yet fully understood. Consequently, it is desirable to provide efficient purification methods for producing the proteins in pure form, which will facilitate studies in vivo and in vitro.
The use of hydroxyapatite, by means of displacement chromatography, has been disclosed in the purification of antithrombin-III (Freitag and Breier (1995) J. Chromatography A, 691, 101-112). However, the purification of separate isoforms of AT-III has not previously been achieved by hydroxyapatite chromatography.